Phthalate Contamination to Vege - ACS Publications - American

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Agricultural and Environmental Chemistry

Bioaugmentation of exogenous strain Rhodococcus sp. 2G can efficiently mitigate DEHP contamination to vegetable cultivation Hai-Ming Zhao, Huan Du, Chun-Qing Huang, Sha Li, Xian-Hong Zeng, Xue-Jing Huang, Lei Xiang, Hui Li, Yan-Wen Li, Quanying Cai, Ce-Hui Mo, and Zhenli He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01875 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

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Journal of Agricultural and Food Chemistry

Bioaugmentation of exogenous strain Rhodococcus sp. 2G can efficiently mitigate DEHP contamination to vegetable cultivation Hai-Ming Zhao†,‡,§, Huan Du†,§, Chun-Qing Huang†, Sha Li†, Xian-Hong Zeng†, Xue-Jing Huang†, Lei Xiang†, Hui Li†, Yan-Wen Li†, Quan-Ying Cai*,†, Ce-Hui Mo*,†, Zhenli He‡

† Guangdong

Provincial Research Center for Environment Pollution Control and Remediation Materials, College

of Life Science and Technology, Jinan University, Guangzhou 510632, China ‡ Indian

River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida,

Fort Pierce, FL 34945, USA

*Correspondence: [email protected] (Q. Cai); [email protected]. (C. Mo); Tel: +86-20-8522-3405 (C. Mo)

1

ACS Paragon Plus Environment


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ABSTRACT: This work developed a bioaugmentation strategy that simultaneously reduced soil

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DEHP pollution and its bioaccumulation in Brassica parachinensis by inoculating the isolated

3

strain Rhodococcus sp. 2G. This strain could efficiently degrade DEHP at a wide concentration

4

range from 50~1600 mg/L and transformed DEHP through a unique biochemical degradation

5

pathway that distinguished it from other Rhodococcus species. Besides, the strain 2G colonized well

6

in the rhizosphere soil of the inoculated vegetable without competition with indigenous microbes,

7

resulting in increased removal of DEHP from soil (~95%) and reduced DEHP bioaccumulation in

8

vegetables (~75% in edible part) synchronously. Improved enzyme activities and DOC content in

9

the rhizosphere of planting vegetable and inoculating strain 2G were responsible for the high

10

efficiency in mitigating DEHP contamination to vegetable cultivation. This work demonstrated a

11

great potential application to grow vegetable in contaminated soil for safe food production.

12

KEYWORDS: phthalate; microbial degradation; bioaugmentation; soil bioremediation;

13

vegetable cultivation

14 15 16 17 18 19 20 21 22 23 2


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1. INTRODUCTION

25

Phthalic acid esters (PAEs) are widely used as plasticizers in plastic products to increase their

26

flexibility and durability.1 They tend to be released into environment and may be ingested by human

27

body through various approaches such as dietary and dermal contact.2,3 Di-(2-ethylhexyl) phthalate

28

(DEHP), classified as the priority pollutant by many governments and regional organizations, one

29

of the PAE compounds widely exists in the various environments.4 It is reported that the suspected

30

endocrine-disrupting DEHP can cause hepatocellular tumors and developmental and reproductive

31

toxicants.5,6 Because of widespread application of plastic film, wastewater irrigation, fertilizers, etc.

32

that usually contain PAEs, elevated concentrations of DEHP were often found in agricultural

33

fields.7-10 DEHP concentration in vegetable soil was reported with up to 57.4 mg/kg in Guangzhou,

34

China.11 DEHP in soil can be taken up and accumulated by crops, especially for leaf

35

vegetables, posing potential harms to the human health through diet.12,13 Our previous investigation

36

suggests that Chinese flowering cabbage (Brassica parachinensis) accumulates the highest level of

37

DEHP (up to 9.3 mg/kg, DW) in all the investigated crops collected from Pearl River Delta area,

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southern China.8 Therefore, it is of great urgency to eliminate PAEs from agricultural soils for safe

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agricultural product.

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Physico-chemical degradation of DEHP in the natural environment is very difficult.14

41

Microbe-mediated biodegradation is the main degradation approach of DEHP, which is the most

42

promising method to remediate DEHP-contaminated environments.15,16 To date, many

43

DEHP-degrading bacteria have been isolated from soils, activated sludge, water, sediment, etc.,

44

with degradation rates of 67-100% within 3-21 days,17-22 but further application potential analysis of

45

these bacteria is poorly studied. Considering the poor nutrients, interspecific competition, and

46

adverse survival conditions (such as unsuitable temperature and pH) in the actual environments, few 3



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of them showed good application potential.15 Moreover, the isolated strains that not only possess

48

high efficiency of DEHP degradation but also ensure complete mineralization are still scarce in

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number, because DEHP has long side chain, high octanol–water partitioning coefficient

50

(log Kow = 7.5), and low water solubility that make it recalcitrant to biodegrade.23 Therefore, it is

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urgent to isolate new DEHP-degrading bacteria for enriching strain resources. Besides, the

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application of the isolates in remediating environment contaminated by DEHP should be widely

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conducted for their real practice.

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Here, an effective DEHP degrading bacterium Rhodococcus sp. 2G was isolated and identified,

55

and its biodegradation characteristics were investigated. Further, pot experiments were conducted to

56

evaluate the interactions between the inoculated strain and the rhizosphere microorganism of B.

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parachinensis, and their effects on the DEHP elimination from contaminated soil and the growing

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vegetable. Meanwhile, the dynamic change of inoculated Rhodococcus sp. 2G in the rhizosphere

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soil was traced by PCR-DGGE analysis.

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2. MATERIALS AND METHODS

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2.1. Culture enrichment and isolation of the PAEs-degrading bacteria. The used

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chemicals and mediums were presented in Supporting Information. The enrichment-culture

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technique was used to isolate effective DEHP degrading bacteria from activated sludges, which

64

were collected from a sewage treatment plant located at Guangzhou, southern China. The initial

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enrichment culture was started by inoculating 100 mL of sterile mineral salt medium (MSM) with 5

66

mL activated sludge suspension and 50 mg/L DEHP in culture flasks (250 mL). These flasks were

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incubated for 7 days at 140 rpm and 30°C in an incubator shaker. Every 7 days the suspension (1

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mL) was transferred to new culture flasks containing fresh MSM (100 mL) supplemented with six 4


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increasing concentrations (100, 200, 300, 600, 1200, and 1500 mg/L) of DEHP, respectively. The

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enrichment medium was diluted serially after six rounds of transfers according to our previous

71

method,19 and then was spread on MSM-agar plate containing DEHP (200 mg/L) as sole carbon

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source for isolating individual colonies. Finally, an isolated strain that could utilize DEHP for

73

growth on MSM, designated as 2G (hereafter, strain 2G), was detailedly characterized in

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Supporting Information.

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2.2. Optimal conditions for DEHP biodegradation. Three main factors including medium

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temperature, pH, and inoculum size were chosen as independent variables based on preliminary

77

single-factor experiment. Table S1 shows the range and center point values of three independent

78

variables. The biodegradation of DEHP (200 mg/L) in MSM for 5 days was as the dependent

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variable. These factors and their interactive influences on DEHP biodegradation by the strain 2G

80

were optimized using response surface methodology (RSM). Statistic Analysis System (SAS)

81

software was used to generate a three-variable Box-Behnken design consisting of 15 experimental

82

runs with three replicates at the midpoint (Table S2). The data were analyzed through response

83

surface regression procedure to fit the following quadratic polynomial equation (Equation 1):

84

𝑌𝑖 = 𝑏0 + ∑𝑏𝑖𝑋𝑖 + ∑𝑏𝑖𝑗𝑋𝑖𝑋𝑗 + ∑𝑏𝑖𝑖𝑋𝑖2

85

Where Yi is the predicted response, Xi and Xj are variables, b0 is the constant, bi is the linear

86

(1)

coefficient, bij is the interaction coefficient, and bii is the quadratic coefficient.

87

The interaction between the variables and the responses was analyzed by analysis of variance

88

(ANOVA) with a 95% confidence level. Three-dimensional response surface was drawn to

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demonstrate individual and interaction effects of the independent variables on the DEHP

90

biodegradation rate.

91

2.3. Preparation of the bacterial suspension. After centrifugation at 4600×g for 10 min, 5



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the degradation bacteria with late-exponential growth phase were collected. The bacteria were

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resuspended in the sterile saline (0.9%) after washing three times with the sterile saline. Unless

94

otherwise stated, the densities of strain 2G were adjusted with the sterile saline to an OD600

95

(optical density measurements at 600 nm) of 0.6. The dilution plate count technique was used to

96

quantify colony forming units (CFU/mL) of the suspension.24 One percent of this suspension was

97

used as the inocula for further study.

nm

98

2.4. Biodegradation of DEHP by the strain 2G at different initial concentrations.

99

Biodegradation experiments were performed in MSM containing DEHP at different initial

100

concentrations (50-1600 mg/L). Under the optimum conditions, the culture mediums inoculated

101

with bacterial suspensions were triplicately incubated for 5 days at 140 rpm, and then were

102

collected daily to determine the residues of DEHP in MSM. The mediums without inoculation was

103

kept as control. Besides, samples of cell-free filtrates at these different initial concentrations of

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DEHP were collected daily to measure cell growth (OD600) using a spectrophotometer, respectively.

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In the meantime, the intermediates of DEHP (200 mg/L) at days 1, 3, and 5 were identified by

106

GC/MS (QP2010 Plus, Shimadzu, Japan).

107

The first-order kinetic equation (2) was used to describe the effect of the initial concentrations of

108

DEHP on biodegrading rate:

109

(2)

𝑙𝑛𝐶 = ―𝑘𝑡 + 𝐴

110

Where C is the DEHP concentration at time t; k and A are the slope and constant of first-order

111

equation, respectively. The equation (3) was used to calculate the theoretical half-life (t1/2) of

112

DEHP: 𝑙𝑛2

(3)

113

𝑡1/2 =

114

Where k is the rate constant (h-1).

𝑘

6


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The substrate inhibition model (Eq. (4)) was used to fit the specific degradation rate (q) at different

116

initial concentrations:25

117

𝑞𝑚𝑎𝑥𝑆

𝑞= (𝑆 +

(4)

( )+𝐾 ) 𝑆2 𝐾𝑖

𝑆

118

Where S and qmax are the substrate concentration and maximum specific growth rate,

119

respectively; KS and Ki are the constants of substrate affinity and inhibition, respectively. The model

120

and kinetics parameters were analyzed using GraphPad Prism software.

121

2.5. Pot experiments. To evaluate the effects of inoculated strain 2G on DEHP residues in soil

122

and vegetable, soil pot experiments were conducted. The soil collected from an agricultural field

123

was obtained by air drying and sieving (2 mm) . The soil physical properties were analyzed (DW)

124

as follows: total organic C, 8.3 g/kg; total P, 1.42 g/kg; total N, 1.31 g/kg; a sandy loam texture

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including silt 53.5%, sand 34.8%, and clay 11.7%; and pH 6.9. Pot experiment was conducted in

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five replicates with four different designs: (i) soil (CK); (ii) soil planted with vegetable (Planting);

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(iii) soil inoculated with Rhodococcus sp. 2G (Inoculation); (iv) soil planted with vegetable and

128

inoculated with Rhodococcus sp. 2G (Planting+Inoculation). All of the soils were spiked with 50

129

mg/kg of DEHP based on the reported highest DEHP concentration in vegetable soil11 and aged for

130

2 weeks prior to use. About 3.0 kg (DW) of the DEHP-spiked soil was loaded into a ceramic pot

131

and mixed with chemical fertilizer (6 g; N:P:K=4:3:4). Five seedlings were transplanted into every

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pot after seed germination, and then the strain 2G was inoculated into the planted soil by drip

133

irrigation. The sterile saline was kept as control and all treatments were performed in triplicate.

134

These pots were kept for 35 days at natural light and moderate moisture conditions in the

135

greenhouse. The rhizosphere soils were collected from the pots, refering to the soils adhering to

136

roots after shaking. All the soil samples were collected at the 7th, 21st, 35th days after transplanting, 7



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respectively. The vegetables were collected at the 35th days and separated into shoots and roots. All

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the samples were stored at −70°C for further analysis.

139

2.6. Instrumental analysis of DEHP and its degradation intermediates. The extraction,

140

clean up, and GC/MS procedures for DEHP and its intermediate analyses were performed based on

141

the methods reported in our previous papers.13,19 The limit of detection (LOD) of DEHP was 2.5

142

μg/L according to three fold of the signal-to-noise ratio. The average procedural blank value (6.3

143

μg/L) was subtracted from each sample. The recoveries of DEHP in soil/plant and filtrate samples

144

ranged from 87.4% to 107.2% and from 97.7% to 104.7%, respectively.

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2.7. Analyses of enzyme activities and DOC in soils. Soil enzyme activities, including

146

dehydrogenase, urease, protease, peroxidase, and polyphenol oxidase (PPO), were determined by a

147

UV-2450 spectrophotometer (Shimadzu, Japan). Briefly, the supernatants for the enzymatic assays

148

were extracted from 5 g of soil samples using 10 mL sterile water. Peroxidase and PPO were

149

assayed based on the methods described in our previous report.20 The detailed description on the

150

methods of dehydrogenase, urease, and protease activities were measured according to previous

151

reports26-28 (see Supporting Information). The control for each enzyme test was carried out without

152

the substrate addition. The concentrations of DOC in soil samples were measured by a TOC-VCSH

153

analyzer (Shimadzu, Japan) according to previous study.20

154

2.8. PCR-DGGE analysis. Total DNA was extracted from 0.5 g of frozen soil samples using

155

the EZNA™ Soil DNA kit (Omega Bio-Tek, USA), and then was used to amplify the short and

156

highly variable V3 region of the bacterial 16S rRNA gene by PCR for the following denaturing

157

gradient gel electrophoresis (DGGE) analysis, using the universal primers 534R and 341F with GC

158

clamp.19 For the DGGE assay, an amount of 25 μL of amplicons was loaded on 10% (w/v)

159

polyacrylamide gels containing a denaturant gradient of 40-60% parallel to electrophoresis direction 8


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made of formamide and urea (100% denaturant contains 7 M urea and 40% formamide). Gels were

161

electrophoresed at constant 60℃ and 80V in 1×TAE buffer (1 mM EDTA, 40 mM Tris-acetate, pH

162

8.5) for 16 h, followed by colouration using 1× SybrGreen I nucleic acid gel stain for 30 min. Bands

163

in digital images were detected by a UVP gel documentation system.

164

2.9. Statistical analysis. The SPSS 17.0 for Windows statistical package was used for these data

165

analyses including standard deviation, regression, and analysis of variance (ANOVA). The analyses

166

were run at least in triplicate and the result is significant at P< 0.05.

167

3. RESULTS AND DISCUSSION

168

3.1. Identification of strain 2G and optimization of culture condition. The strain 2G

169

was identified as Rhodococcus sp. and its detailed characterization was presented in Supporting

170

Information. The effects of important variables containing pH (X1), temperature (X2), and inoculums

171

size (X3) on DEHP biodegradation were determined by the Box-Behnken design according to

172

previous single-factor experiments. Table S2 shows the related experimental design and response

173

for DEHP biodegradation. The second order polynomial (regression) equation was used to represent

174

the response surface Y as follows (5):

175 176 177

𝑌2𝐺 = 98.3 ― 3𝑋1 +2.238𝑋2 ―10.252𝑋12 ―5.777𝑋22

(5)

Where Y2G is the predicted DEHP biodegradation (%) by strain 2G, and X1 and X2 are the coded values for the pH and temperature, respectively.

178

As shown in Table 1, the determination coefficient R2 of the model is 0.9698, which is very

179

close to 1. The result suggested that approximately 97% variation can be explained by this model,

180

indication of good accuracy. The regression parameters in Table S4 indicated that the linear and

181

square terms of pH (X1) and temperature (X2) values had significant effects (P0.05). Hence, the three-dimensional (3D) plot displayed the effects of temperature

184

and pH on DEHP biodegradation while the inoculum size was fixed at 0.6 (OD600nm) (Figure 1).

185

The theoretical maximum value of DEHP biodegradation rate was 98.1%. At the theoretical

186

maximum point, the optimal conditions for DEHP biodegradation by strain 2G were pH 7.1,

187

temperature 29℃, and inoculum size 0.6 (OD600nm).

188

3.2. Strain 2G growth and DEHP biodegradation in MSM. Strain 2G could utilize DEHP

189

to grow rapidly without lag phases (Figure S3), reflecting rapid adaptation to DEHP at various

190

initial concentrations. At low concentrations of DEHP (≤400 mg/L), strain 2G could grow rapidly

191

and the cell densities increased to their maximum levels. While the initial concentrations increased

192

from 800 to 1600 mg/L, the growing trends of strain 2G showed continuous rising within 5 days.

193

Overall, strain 2G could degrade DEHP rapidly at the beginning of the incubation period without an

194

adjustment process, further indicating its great application potential to remediate the

195

DEHP-contaminated environment.

196

As shown in Figure 2a, strain 2G could rapidly degrade up to 1600 mg/L of DEHP, with almost

197

complete degradation at low concentrations (50-800 mg/L) within 5 days. The degradation rate kept

198

higher when the DEHP concentration increased to 1600 mg/L, with 80% of the removal rate within

199

5 days even initial DEHP concentration was up to 1600 mg/L. Inhibition was also found with

200

increasing concentration of xenobiotics during the degradation by other bacteria.17,21

201

Table 2 shows the kinetic parameters for different initial concentrations of DEHP. The R2 values

202

were all higher than 0.9, indication of a good mathematical fit to the first-order kinetics model. It is

203

concluded that the initial concentrations had critical impact on the biodegradation efficiency of

204

DEHP due to the distinct difference in half-life (t1/2) of DEHP (varying from 0.86 to 2.53 days). To

205

further clarify the impacts of initial concentrations of DEHP on the biodegradation, the specific 10


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degradation rate (q) was calculated based on the substrate inhibition model (Eq. (4) (Figure 2b). The

207

coefficient of determination R2 (0.981) indicated that the experimental data agreed well with this

208

model. The kinetic parameters of strain 2G determined from the non-linear regression analysis

209

according to Chen et al.25 were qmax of 1.159 day-1, Ks of 213.3 mg/L, and Ki of 531.6 mg/L. The Ks

210

and Ki numerically equal the lowest and the highest concentrations of substrate where the specific

211

growth rates are equal to one-half the maximum specific growth rates in the absence of inhibition,

212

respectively, which are important parameters in understanding the kinetics of the microorganism in

213

the system.29,30 Both the two parameters were higher than those of the DEHP-degrading strain

214

Microbacterium sp. J-1 (Ks of 180.2 mg/L and Ki of 332.8 mg/L),20 indicating a better substrate

215

adaptability for strain 2G. Additionally, the maximum inhibitor concentration (Sm) was calculated to

216

be 336.62 mg/L based on the square root of Ki×Ks,29 indicating that this concentration could

217

partially inhibit the activity of strain 2G to degrade DEHP. However, it is hard to happen in the case

218

of natural environments because the reported highest DEHP concentrations whether in water (≤

219

0.36 mg/L), sewage (≤ 154 mg/kg) or in soil (≤ 149 mg/kg)11,31,32 are much less than this value of

220

Sm. Therefore, it is expected that the strain 2G can work well in bioremediation of

221

DEHP-contaminated environment.

222

3.3. Biochemical degradation pathway of DEHP by strain 2G. To study the biochemical

223

degradation pathway of DEHP by strain 2G, the degradation intermediates in culture filtrates were

224

extracted and identified by GC/MS. After 3 days of culture, five distinct peaks were observed, and

225

the corresponding retention times (RT), predicted chemical structures, characteristic ions of the

226

mass spectra (m/z) are given in Table S5. At the 3rd day of the degradation experiment, five

227

chromatographic peaks were found at the retention time of 10.05, 9.56, 6.89, 6.31, and 5.62 min,

228

respectively (Figure 3), while only one peak at 10.05 min was found before the experiment. The 11



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five peaks were identified as DEHP, MEHP, PA, catechol and BA, respectively, based on their

230

retention times and most typical fragment ions (Table S5). Afterwards, these peaks decreased

231

gradually and disappeared finally with continuous culture. No persistent accumulative metabolites

232

were detected in the final experiment. It was supported by our previous potential intermediate

233

utilization tests that the strain 2G was able to live well on these intermediates as sole sources of

234

carbon and energy.36

235

It is reported that the primary metabolic pathways of PAEs include two steps: (i) transformation

236

of PAEs to PA, and (ii) utilization of PA.15 In general, two kinds of reactions were involved in

237

transforming PAEs to PA: the one is reduction of side chains’ length by β-oxidation or

238

trans-esterification, and the other is hydrolyzing of ester bonds.16 In the present study, DEHP might

239

be hydrolyzed by esterase firstly to MEHP and then further to PA based on the identified

240

intermediates. PA is the central intermediate of reported PAEs biodegrading process, and its

241

metabolic pathways are systematically reviewed elsewhere.15,16 Under aerobic condition, the key

242

step in the biochemical degradation of PA is the hydroxylation of the aromatic ring by phthalate

243

dioxygenase to form the common intermediate PCA.15,37 In this study, however, the

244

chromatographic peak of PCA was not found in the identified DEHP degradation intermediates.

245

Consistently, no gene involved in PA degrading to PCA was found based on the genome

246

sequencing of strain 2G.36 Alternately, based on the detection of BA and catechol, it is supposed

247

that Rhodococcus sp. 2G might metabolize PA to BA in priority through decarboxylation, and

248

followed by ring hydroxylation and opening to form catechol, which was further utilized in

249

tricarboxylic acid (TCA) cycle. Based on our previous results of functional genomic analysis, the

250

identified genes, including transferase/decarboxylase genes, Xyl gene cluster, Pca-Cat gene cluster,

251

Icl-Mhp gene cluster, etc., might be involved in the above pathway.36 Overall, a possible pathway 12


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252

for DEHP biochemical degradation by Rhodococcus sp. 2G was proposed based on the results and

253

related chemical properties (Figure 3): DEHP was transformed to MEHP and PA by hydrolysis, and

254

then BA was formed through the decarbxylation of PA; catechol was further produced through the

255

corresponding hydroxylation and decarboxylation of BA; finally, the terminal degradation products

256

of CO2 and H2O occurred after the generated catechol entering the TCA cycle. The detailed

257

enzymatic steps and gene clusters involved in this biochemical degradation pathway were

258

evidenced by functional genomic analysis of strain 2G previously.36 Taken together, this is the first

259

time to comprehensively and systematically unravel such a unique biochemical degradation

260

pathway of DEHP in a Rhodococcus species, which distinguishes strain 2G from other

261

Rhodococcus species.

262

3.4. Effect of the inoculated 2G on decontamination of DEHP-contaminated soil

263

and vegetable. The microbial inoculation of strain 2G in DEHP-contaminated soils with or

264

without planting vegetable was conducted to explore the impact of inoculated strain 2G on

265

mitigating DEHP contamination to vegetable cultivation (Figure 4). The results showed that the

266

introduction of strain 2G as a bioaugmentation strategy could efficiently enhance the degradation of

267

DEHP in contaminated soil after 35 days, with removal rate (80%) significantly higher (P

Phthalate Contamination to Vege - ACS Publications - American

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